This invention relates to compositions and methods for modulating expression of the E2A-Pbx1 gene which results from the fusion of the E2A and Pbx1 genes. These methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of conditions associated with expression of the E2A-Pbx1 gene.
Genetic alterations are responsible for a multitude of cancers. One type of alteration is chromosomal translocation where different chromosomes are fused or where different regions within a single chromosome are fused. Both random and distinctive rearrangements can occur, with random rearrangements being common in solid tumors, while distinctive rearrangements are common in leukemias, lymphomas and sarcomas (Lengauer C., et al., Nature, 1998, 396, 643-649). These distinctive rearrangements involve the same breakpoints and often occur within transcription factors. The resulting transcripts can be targeted using sequence specific approaches.
Many acute leukemias are characterized by chromosomal translocations that involve transcription factors. In pediatric acute lymphoblastoid leukemia (ALL) carrying the t(1;19) chromosomal translocation, the N-terminal transactivation domain of the E2A transcription factor is fused to the homeodomain of Pbx-1 (Prl) to create the chimeric transcription factor, E2A-Pbx1 (Kamps, M. P., Curr. Top. Microbiol. Immunol., 1997, 220, 25-43). This particular translocation has been found in 23% of pediatric pre-B ALL (Carroll, A., et al., Blood, 1984, 63, 721-724) and is associated with a poorer prognosis (Crist, W., et al., Blood, 1990, 76, 117-122). The resulting chimeric protein has shown transforming ability in NIH3T3 cells (Kamps, M. P., et al., Genes Dev., 1991, 5, 358-368), the ability to induce malignant lymphomas in transgenic mice (Dedera, D. A., et al., Cell, 1993, 74, 833-843) and capable of promoting the rapid development of myeloid leukemias (Kamps, M. P. and Baltimore, D., Mol. Cell. Biol., 1993, 13, 351-357).
E2A-Pbx1 physically interacts with homeodomain proteins, which are effectors of differentiation. In particular, Pbx1 interacts with Hox proteins, which are also transcription factors. Overexpression of Hox proteins can induce transformation. Pbx1 is an important contributor to differentiation, via its interaction with Hox proteins.
Expression of E2A-Pbx1 is involved in development of pre-B-cell acute lymphocytic leukemia. Expression of this protein prevents the differentiation of pre-B-cells and promotes their growth. One of the genes upregulated by E2A-Pbx-1is granulocyte colony-stimulating factor receptor (de Lau, W. B. M., et al., Oncogene, 1998, 17, 503-510).
Chemotherapy, including the use of methotrexate, and bone marrow transplants are the most effective treatments for leukemia and are not specific for E2A-Pbx1. There is a lack of specific inhibitors of E2A-Pbx1.
There remains a need for improved compositions and methods for inhibiting E2A-Pbx1 gene expression.
The present invention provides antisense compounds which are targeted to nucleic acids encoding E2A-Pbx1 (E2A-prl) and are capable of modulating E2A-Pbx1 mediated transformation. In one embodiment, the present invention provides oligonucleotides, including chimeric oligonucleotides, targeted to nucleic acids encoding E2A-Pbx1. The compounds of the invention are believed to be useful both diagnostically and therapeutically, and are believed to be particularly useful in the methods of the present invention.
The present invention also comprises methods of modulating E2A-Pbx1 mediated transformation, in cells and tissues, using the antisense compounds of the invention. Methods of inhibiting E2A-Pbx1 expression are provided; these methods are believed to be useful both therapeutically and diagnostically. These methods are also useful as tools, for example, for detecting and determining the role of E2A-Pbx1 in various cell functions and physiological processes and conditions and for diagnosing conditions associated with expression of E2A-Pbx1.
The present invention also comprises methods for diagnosing and treating cancers, especially pre-B-cell acute lymphocytic leukemia. These methods are believed to be useful, for example, in diagnosing E2A-Pbx1-associated disease progression. Furthermore, these methods are believed to be useful both therapeutically, including prophylactically, and as clinical research and diagnostic tools.
Thus, in a first aspect the present invention features an antisense compound having at least one modified internucleoside linkage, targeted to E2A-Pbx1 mRNA, and which inhibits the expression of the E2A-Pbx1 gene product.
In a preferred embodiment the antisense compound is an olilgonucleotide and is between 8 to 30 nucleobases in length, more preferably the antisense compound is up to 20 bases, and most preferably it is 15 to 20 bases long. In a further embodiment the modified internucleoside linkage is a phosphorothioate linkage and the nucleotide comprises a modified sugar moiety such as for example a 2xe2x80x2-O-methoxyethyl moiety, and/or a 5-methyl cytosine.
E2A-Pbx1 is the resulting product of the N-terminal transactivation domain of the E2A transcription factor becoming fused to the homeodomain of Pbx-1 (Prl). Thus in a preferred embodiment, the present invention features an antisense compound targeted to E2A and/or Pbx1, more preferably the antisense compound is target to bases 388-1812 of homo sapiens pre-B-cell leukemia transcription factor 1 (PBX1) mRNA (Genbank Accession No. NM002585) and/or bases 853-14801 of human transcription factor (E2A) mRNA (Genbank Accession number M31523). In another preferred embodiment the antisense compound is targeted to the fusion junction of E2A-Pbx1. It is also preferred that the antisense compound be contained within a pharmaceutical composition.
In an additional aspect the present invention features methods of inhibiting the expression of E2A-Pbx1 in cells or tissues. Within this method the cells or tissues are contacted with the antisense compound presently described so that expression of E2A-Pbx1 is inhibited. It is preferred that the cells or tissues are human.
In a further aspect the invention features methods for treating a human subject having a disease or condition associated with E2A-Pbx1 by administering to the subject a therapeutically or prophylactically effective amount of the antisense compound as described herein so that expression of E2A-Pbx1 is inhibited. In a preferred embodiment the disease or condition is, for example, pre-B-cell acute lymphocytic leukemia, and/or sarcomatous cancer.
Further aspects of the invention are described within the description of the preferred embodiments. The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention and from the claims.
E2A-Pbx1 plays an important role in the development of leukemias, lymphomas and sarcomas. Expression of this gene is associated with a poor prognosis in pre-B-cell acute lymphocytic leukemia. As such, this chimeric protein represents an attractive target for treatment of such diseases. In particular, modulation of the expression of E2A-Pbx1 may be useful for the treatment of pre-B-cell acute lymphocytic leukemia.
The cDNA sequence of human E2A-Pbx1 (PRL) is available via Genbank Accession No. M31522. In accordance with Kamps et al. (supra) comparison of the sequence with the individual sequences for E2A and Pbx1 shows the fusion/junction point of the two genes to be at nucleotide 625 of Genbank Accession No. M31522. In a preferred embodiment the antisense compounds as described herein contain oligonucleotides targeted to the junction in E2A-Pbx1, such as for example oligonucleotides 154015, 15407, and 15409, SEQ ID NOS: 10, 11 and 12 respectively. Furthermore, due to the fusion of E2A to Pbx1, also included in the present invention are antisense compounds containing oligonucleotides targeted to E2A and/or Pbx1.
E2A, an Ig regulator, is normally expressed, while Pbx1 expression is thought to be only expressed embryonically. Therefore in a preferred embodiment the Pbx portion of the fusion is targeted. Since Pbx is only expressed in the fusion product, the aberrant E2A-Pbx mRNA would be reduced/without affecting normally expressed genes. Therefore in an additional embodiment, the invention described herein provides antisense compounds targeted to Pbx1.
The present invention employs antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding E2A-Pbx1, ultimately modulating the amount of E2A-Pbx1 produced. This is accomplished by providing oligonucleotides which specifically hybridize with nucleic acids, preferably mRNA, encoding E2A-Pbx1.
This relationship between an antisense compound such as an oligonucleotide and its complementary nucleic acid target, to which it hybridizes, is commonly referred to as xe2x80x9cantisensexe2x80x9d. xe2x80x9cTargetingxe2x80x9d an oligonucleotide to a chosen nucleic acid target, in the context of this invention, is a multistep process. The process usually begins with identifying a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA made from the gene) whose expression is associated with a particular disease state, or a nucleic acid from an infectious agent. In the present invention, the targets are nucleic acids encoding E2A-Pbx1; in other words, a gene encoding E2A/Pbx1 fusion protein, or mRNA expressed from the E2A-Pbx1 genes. mRNA which encodes E2A-Pbx1 is presently the preferred target. The targeting process also includes determination of a site or sites within the nucleic acid sequence for the antisense interaction to occur such that modulation of gene expression will result.
In accordance with this invention, persons of ordinary skill in the art will understand that messenger RNA includes not only the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5xe2x80x2-untranslated region, the 3xe2x80x2-untranslated region, the 5xe2x80x2 cap region and intron/exon junction ribonucleotides. Thus, oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides. The oligonucleotide may therefore be specifically hybridizable with a transcription initiation site region, a translation initiation codon region, a 5xe2x80x2 cap region, an intron/exon junction, coding sequences, a translation termination codon region or sequences in the 5xe2x80x2- or 3xe2x80x2-untranslated region. Since, as is known in the art, the translation initiation codon is typically 5xe2x80x2-AUG (in transcribed mRNA molecules; 5xe2x80x2-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the xe2x80x9cAUG codon,xe2x80x9d the xe2x80x9cstart codonxe2x80x9d or the xe2x80x9cAUG start codon.xe2x80x9d A minority of genes have a translation initiation codon having the RNA sequence 5xe2x80x2-GUG, 5xe2x80x2-UUG or 5xe2x80x2-CUG, and 5xe2x80x2-AUA, 5xe2x80x2-ACG and 5xe2x80x2-CUG have been shown to function in vivo. Thus, the terms xe2x80x9ctranslation initiation codonxe2x80x9d and xe2x80x9cstart codonxe2x80x9d can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, xe2x80x9cstart codonxe2x80x9d and xe2x80x9ctranslation initiation codonxe2x80x9d refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding E2A-Pbx1, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or xe2x80x9cstop codonxe2x80x9d) of a gene may have one of three sequences, i.e., 5xe2x80x2-UAA, 5xe2x80x2-UAG and 5xe2x80x2-UGA (the corresponding DNA sequences are 5xe2x80x2-TAA, 5xe2x80x2-TAG and 5xe2x80x2-TGA, respectively). The terms xe2x80x9cstart codon region,xe2x80x9d xe2x80x9cAUG regionxe2x80x9d and xe2x80x9ctranslation initiation codon regionxe2x80x9d refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5xe2x80x2 or 3xe2x80x2) from a translation initiation codon. This region is a preferred target region. Similarly, the terms xe2x80x9cstop codon regionxe2x80x9d and xe2x80x9ctranslation termination codon regionxe2x80x9d refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5xe2x80x2 or 3xe2x80x2) from a translation termination codon. This region is a preferred target region. The open reading frame (ORF) or xe2x80x9ccoding region,xe2x80x9d which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other preferred target regions include the 5xe2x80x2 untranslated region (5xe2x80x2UTR), known in the art to refer to the portion of an mRNA in the 5xe2x80x2 direction from the translation initiation codon, and thus including nucleotides between the 5xe2x80x2 cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene and the 3xe2x80x2 untranslated region (3xe2x80x2UTR), known in the art to refer to the portion of an mRNA in the 3xe2x80x2 direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3xe2x80x2 end of an mRNA or corresponding nucleotides on the gene. The 5xe2x80x2 cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5xe2x80x2-most residue of the mRNA via a 5xe2x80x2-5xe2x80x2 triphosphate linkage. The 5xe2x80x2 cap region of an mRNA is considered to include the 5xe2x80x2 cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5xe2x80x2 cap region may also be a preferred target region.
Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as xe2x80x9cintronsxe2x80x9d, which are excised from a pre-mRNA transcript to yield one or more mature mRNA. The remaining (and therefore translated) regions are known as xe2x80x9cexonsxe2x80x9d and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., exon-exon or intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. Targeting particular exons in alternatively spliced mRNAs may also be preferred. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.
Once the target site or sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired modulation.
xe2x80x9cHybridizationxe2x80x9d, in the context of this invention, means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them.
xe2x80x9cSpecifically hybridizablexe2x80x9d and xe2x80x9ccomplementaryxe2x80x9d are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide.
It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment and, in the case of in vitro assays, under conditions in which the assays are conducted.
Hybridization of antisense oligonucleotides with mRNA interferes with one or more of the normal functions of mRNA. The functions of mRNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA.
The overall effect of interference with mRNA function is modulation of expression of E2A-Pbx1. In the context of this invention xe2x80x9cmodulationxe2x80x9d means either inhibition or stimulation; i.e., either a decrease or increase in expression. This modulation can be measured in ways which are routine in the art, for example by Northern blot assay of mRNA expression, reverse transcriptase PCR, Western blot or ELISA assay of protein expression, or by an immunoprecipitation assay of protein expression. Effects on cell proliferation or tumor cell growth can also be measured using methods well known in the art. Inhibition is presently the preferred form of modulation.
The oligonucleotides of this invention can be used in diagnostics, therapeutics, prophylaxis, and as research reagents and in kits. Since the oligonucleotides of this invention hybridize to nucleic acids encoding E2A-Pbx1, sandwich, calorimetric and other assays can easily be constructed to exploit this fact. Provision of means for detecting hybridization of oligonucleotide with the E2A-Pbx1 genes or mRNA can routinely be accomplished. Such provision may include enzyme conjugation, radiolabelling or any other suitable detection systems. Kits for detecting the presence or absence of E2A-Pbx1 may also be prepared.
The present invention is also suitable for diagnosing genetic alterations responsible for disease states such as cancer, in particular, in patients suspected of having leukemia, such as pediatric pre-B acute lymphoblastoid leukemia. A number of assays may be formulated employing the present invention, which assays will commonly comprise contacting a tissue sample with an oligonucleotide of the invention under conditions selected to permit detection of the alterations and, usually, quantitation. In the context of this invention, to xe2x80x9ccontactxe2x80x9d tissues or cells or cultures of tissues or cells with an oligonucleotide or oligonucleotides means to add the oligonucleotide(s), usually in a liquid carrier, to a cell suspension or tissue sample, either in vitro or ex vivo, or to administer the oligonucleotide(s) to cells or tissues within an animal or a human subject.
By use of term xe2x80x9cculturexe2x80x9d is meant the propagation of cells. Various culture methods exist and are included within the scope of the invention, methods such as, but not limited to, tissue culture methods, batch culture methods, enrichment culture methods, and ex vivo culture methods. In all culture methods the cells to be propagated should be in a nutritive environment which allows for continued cell growth. Tissue and cell culture methods are well understood in the art as these methods have been regularly practiced in various scientific fields for years. Such cultures may be propagated in natural serum or in artificial serum as described for example in U.S. Pat. No. 4,657,866 to Kumar, Sudhir. Inasmuch as a culture represents a group of cells being observed for the presence of a genetic alteration and the determination of a cancer state such as pre-B acute lymphoblastoid leukemia, included within the scope of the invention are cultures of cells on or in a host, such as a tumor which must remain on or in the host to be propagated and treated.
The oligonucleotides of this invention may also be used for research purposes. Thus, the specific hybridization exhibited by the oligonucleotides may be used for assays, purifications, cellular product preparations and in other methodologies which may be appreciated by persons of ordinary skill in the art.
In the context of this invention, the term xe2x80x9colgonucleotidexe2x80x9d refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intersugar (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced binding to target and increased stability in the presence of nucleases.
The antisense compounds in accordance with this invention preferably comprise from about 5 to about 50 nucleobases. Particularly preferred are antisense oligonucleotides comprising from about 8 to about 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides). As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2xe2x80x2, 3xe2x80x2 or 5xe2x80x2 hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3xe2x80x2 to 5xe2x80x2 phosphodiester linkage.
Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3xe2x80x2-alkyene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3xe2x80x2-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3xe2x80x2-5xe2x80x2 linkages, 2xe2x80x2-5xe2x80x2 linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3xe2x80x2-5xe2x80x2 to 5xe2x80x2-3xe2x80x2 or 2xe2x80x2-5xe2x80x2 to 5xe2x80x2-2xe2x80x2. Various salts, mixed salts and free acid forms are also included.
Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863, 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.
Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.
In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNA compounds can be found in Nielsen et al. (Science, 1991, 254, 1497-1500).
Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular xe2x80x94CH2xe2x80x94NHxe2x80x94Oxe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94N(CH3)xe2x80x94Oxe2x80x94CH2xe2x80x94 [known as a methylene (methylimino) or MMI backbone], xe2x80x94CH2xe2x80x94Oxe2x80x94N(CH3)xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94N(CH3)xe2x80x94N(CH3)xe2x80x94CH2xe2x80x94 and xe2x80x94Oxe2x80x94N(CH )3xe2x80x94CHxe2x80x942CHxe2x80x942 [wherein the native phosphodiester backbone is represented as xe2x80x94Oxe2x80x94Pxe2x80x94Oxe2x80x94CH2xe2x80x94] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2xe2x80x2 position: OH; F; Oxe2x80x94, Sxe2x80x94, or N-alkyl, O-alkyl-O-alkyl, Oxe2x80x94, Sxe2x80x94, or N-alkenyl, or Oxe2x80x94, Sxe2x80x94 or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH12)nOCH3, O(CH2)2ON(CH3)2, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2xe2x80x2 position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl; aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2xe2x80x2-methoxyethoxy (2xe2x80x2-Oxe2x80x94CH2CH2OCH3, also known as 2xe2x80x2-O-(2-methoxyethyl) or 2xe2x80x2-MOE) (Martin et al., Helv. Chim. Acta 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2xe2x80x2-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2xe2x80x2-DMAOE, and 2xe2x80x2-dimethylamino-ethoxyethoxy (2xe2x80x2-DMAEOE), i.e., 2xe2x80x2-Oxe2x80x94CH2xe2x80x94Oxe2x80x94CH2xe2x80x94N(CH2)2.
Other preferred modifications include 2xe2x80x2-methoxy (2xe2x80x2-Oxe2x80x94CH3), 2xe2x80x2-aminopropoxy (2xe2x80x2-OCH2CH2CH2NH2) and 2xe2x80x2-fluoro (2xe2x80x2-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3xe2x80x2 position of the sugar on the 3xe2x80x2 terminal nucleotide or in 2xe2x80x2-5xe2x80x2 linked oligonucleotides and the 5xe2x80x2 position of 5xe2x80x2 terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugars structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920.
Oligonucleotides may also include nucleobase (often referred to in the art simply as xe2x80x9cbasexe2x80x9d) modifications or substitutions. As used herein, xe2x80x9cunmodifiedxe2x80x9d or xe2x80x9cnaturalxe2x80x9d nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C or m5c), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering 1990, pages 858-859, Kroschwitz, J. I., ed. John Wiley and Sons, those disclosed by Englisch et al. (Angewandte Chemie, international Edition 1991, 30, 613-722), and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications 1993, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azpyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2xc2x0 C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications 1993, CRC Press, Boca Raton, pages 276-278) and are presently preferred base substitutions, even more particularly when combined with 2xe2x80x2-O-methoxyethyl sugar modifications.
Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos: 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; and 5,681,941.
Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett. 1994, 4, 1053-1059), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci. 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let. 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J. 1991, 10, 1111-1118; Kabanov et al., FEBS Lett. 1990, 259, 327-330; Svinarchuk et al., Biochimie 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett. 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res. 1990, 18, 3777-3783), a polyamine or a polyethylene glylol chain (Manoharan et al., Nucleosides and Nucleotides 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett. 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. 1996, 277, 923-937).
Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 5,472,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.
The present invention also includes oligonucleotides which are chimeric oligonucleotides. xe2x80x9cChimericxe2x80x9d oligonucleotides or xe2x80x9cchimeras,xe2x80x9d in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense inhibition of gene expression. Cleavage of the RNA target can be routinely detected by gel electrophoresis and if necessary, associated nucleic acid hybridization techniques known in the art. This RNAse H-mediated cleavage of the RNA target is distinct from the use of ribozymes to cleave nucleic acids. Ribozymes are not comprehended by the present invention.
Examples of chimeric oligonucleotides include but are not limited to xe2x80x9cgapmers,xe2x80x9d in which three distinct regions are present, normally with a central region flanked by two regions which are chemically equivalent to each other but distinct from the gap. A preferred example of a gapmer is an oligonucleotide in which a central portion (the xe2x80x9cgapxe2x80x9d) of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2xe2x80x2-deoxynucleotides, while the flanking portions (the 5xe2x80x2 and 3xe2x80x2 xe2x80x9cwingsxe2x80x9d) are modified to have greater affinity for the target RNA molecule but are unable to support nuclease activity (e.g., fluoro- or 2xe2x80x2-O-methoxyethyl-substituted). Chimeric oligonucleotides are not limited to those with modifications on the sugar, but may also include oligonucleosides or oligonucleotides with modified backbones, e.g., with regions of phosphorothioate (Pxe2x95x90S). and phosphodiester (Pxe2x95x90O) backbone linkages or with regions of MMI and Pxe2x95x90S backbone linkages. Other chimeras include xe2x80x9cwingmers,xe2x80x9d also known in the art as xe2x80x9chemimers,xe2x80x9d that is, oligonucleotides with two distinct regions. In a preferred example of a wingmer, the 5xe2x80x2 portion of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2xe2x80x2-deoxynucleotides, whereas the 3xe2x80x2 portion is modified in such a fashion so as to have greater affinity for the target RNA molecule but is unable to support nuclease activity (e.g., 2xe2x80x2-fluoro- or 2xe2x80x2-O-methoxyethyl-substituted), or vice-versa. In one embodiment, the oligonucleotides of the present invention contain a 2xe2x80x2-O-methoxyethyl (2xe2x80x2-Oxe2x80x94CH2CH2OCH3) modification on the sugar moiety of at least one nucleotide. This modification has been shown to increase both affinity of the oligonucleotide for its target and nuclease resistance of the oligonucleotide. According to the invention, one, a plurality, or all of the nucleotide subunits of the oligonucleotides of the invention may bear a 2xe2x80x2-O-methoxyethyl (xe2x80x94Oxe2x80x94CH2CH2OCH3) modification. Oligonucleotides comprising a plurality of nucleotide subunits having a 2xe2x80x2-O-methoxyethyl modification can have such a modification on any of the nucleotide subunits within the oligonucleotide, and may be chimeric oligonucleotides. Aside from or in addition to 2xe2x80x2-O-methoxyethyl modifications, oligonucleotides containing other modifications which enhance antisense efficacy, potency or target affinity are also preferred. Chimeric oligonucleotides comprising one or more such modifications are presently preferred.
The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of the routineer. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and 2xe2x80x2-alkoxy or 2xe2x80x2-alkoxyalkoxy derivatives, including 2xe2x80x2-O-methoxyethyl oligonucleotides (Martin, P., Helv. Chim. Acta 1995, 78, 486-504). It is also wells known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and/or CPG (available from Glen Research, Sterling, Va.) to synthesize fluorescently labeled, biotinylated or other conjugated oligonucleotides.
The antisense compounds of the present invention include bioequivalent compounds, including pharmaceutically acceptable salts and prodrugs. This is intended to encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of the nucleic acids of the invention and prodrugs of such nucleic acids. A pharmaceutically acceptable salts@ are physiologically and pharmaceutically acceptable salts of the nucleic acids of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto (see, for example, Berge et al., xe2x80x9cPharmaceutical Salts,xe2x80x9d J. of Pharma Sci. 1977, 66, 1-19).
For oligonucleotides, examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.
The oligonucleotides of the invention may additionally or alternatively be prepared to be delivered in a Aprodrug@ form. The term Aprodrug@ indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl)phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993.
For therapeutic or prophylactic treatment, oligonucleotides are administered in accordance with this invention. Oligonucleotide compounds of the invention may be formulated in a pharmaceutical composition, which may include pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients and the like in addition to the oligonucleotide. Such compositions and formulations are comprehended by the present invention.
Pharmaceutical compositions comprising the oligonucleotides of the present invention may include penetration enhancers in order to enhance the alimentary delivery of the oligonucleotides. Penetration enhancers may be classified as belonging to one of five broad categories, i.e. fatty acids, bile salts, chelating agents, surfactants and non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, 8, 91-192; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1-33). One or more penetration enhancers from one or more of these broad categories may be included.
Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acids, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a. 1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines mono- and di-glycerides and physiologically acceptable salts thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1; El-Hariri et al., J. Pharm. Pharmacol. 1992 44, 651-654).
The physiological roles of bile include the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman and Gilman""s The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term xe2x80x9cbile saltxe2x80x9d includes any of the naturally occurring components of bile as well as any of their synthetic derivatives.
Complex formulations comprising one or more penetration enhancers may be used. For example, bile salts may be used in combination with fatty acids to make complex formulations.
Chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) [Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1-33; Buur et al., J. Control Rel. 1990, 14, 43-51). Chelating agents have the added advantage of also serving as DNase inhibitors.
Surfactants include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al., J. Pharm. Phamacol. 1988, 40, 252-257).
Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol. 1987, 39, 621-626).
As used herein, xe2x80x9ccarrier compoundxe2x80x9d refers to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in at substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. In contrast to a carrier compound, a xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d (excipient) is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The pharmaceutically acceptable carrier may be liquid or solid and is selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinyl-pyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodium starch glycolate, etc.); or wetting agents (e.g., sodium lauryl sulphate, etc.). Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are described in U.S. Pat. Nos. 4,704,295; 4,556,552; 4,309,406; and 4,309,404.
The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional compatible pharmaceutically-active materials such as, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the invention.
Regardless of the method by which the oligonucleotides of the invention are introduced into a patient, colloidal dispersion systems may be used as delivery vehicles to enhance the in vivo stability of the oligonucleotides and/or to target the oligonucleotides to a particular organ, tissue or cell type. Colloidal dispersion systems include, but are not limited to, macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid:oligonucleotide complexes of uncharacterized structure. A preferred colloidal dispersion system is a plurality of liposomes. Liposomes are microscopic spheres having an aqueous core surrounded by one or more outer layers made up of lipids arranged in a bilayer configuration (see, generally, Chonn et al., Current Op. Biotech. 1995, 6, 698-708).
The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, intradermal, epidermal, and transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration, e.g., by inhalation or insufflation, or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2xe2x80x2-O-ethoxyethyl modification are believed to be particularly useful for oral administration.
Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.
Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
Compositions for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. In some cases it may be more effective to treat a patient with an oligonucleotide of the invention in conjunction with other traditional therapeutic modalities in order to increase the efficacy of a treatment regimen. In the context of the invention, the term xe2x80x9ctreatment regimenxe2x80x9d is meant to encompass therapeutic, palliative and prophylactic modalities. For example, a patient may be treated with conventional chemotherapeutic agents, particularly those used for tumor and cancer treatment. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA) 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987 pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such, chemotherapeutic. agents, (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide).
The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in vitro and in in vivo animal models. In general, dosage is from 0.01 xcexcg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 xcexcg to 100 g per kg of body weight, once or more daily, to once every 20 years.
The following examples illustrate the present invention and are not intended to limit the same.